Vitamin C attenuates predisposition to high-fat diet-induced metabolic dysregulation in GLUT10-deficient mouse model

Abstract

Background: The development of type 2 diabetes mellitus (T2DM) is highly influenced by complex interactions between genetic and environmental (dietary and lifestyle) factors. While vitamin C (ascorbic acid, AA) has been suggested as a complementary nutritional treatment for T2DM, evidence for the significance and beneficial effects of AA in T2DM is thus far inconclusive. We suspect that clinical studies on the topic might need to account for combination of genetic and dietary factors that could influence AA effects on metabolism. In this study, we tested this general idea using a mouse model with genetic predisposition to diet-induced metabolic dysfunction. In particular, we utilized mice carrying a human orthologous GLUT10^G128E variant (GLUT10^G128E mice), which are highly sensitive to high-fat diet (HFD)-induced metabolic dysregulation. The genetic variant has high relevance to human populations, as genetic polymorphisms in glucose transporter 10 (GLUT10) are associated with a T2DM intermediate phenotype in nondiabetic population.

Results: We investigated the impacts of AA supplementation on metabolism in wild-type (WT) mice and GLUT10^G128E mice fed with a normal diet or HFD. Overall, the beneficial effects of AA on metabolism were greater in HFD-fed GLUT10^G128E mice than in HFD-fed WT mice. At early postnatal stages, AA improved the development of compromised epididymal white adipose tissue (eWAT) in GLUT10^G128E mice. In adult animals, AA supplementation attenuated the predisposition of GLUT10^G128E mice to HFD-triggered eWAT inflammation, adipokine dysregulation, ectopic fatty acid accumulation, metabolic dysregulation, and body weight gain, as compared with WT mice.

Conclusions: Taken together, our findings suggest that AA has greater beneficial effects on metabolism in HFD-fed GLUT10^G128E mice than HFD-fed WT mice. As such, AA plays an important role in supporting eWAT development and attenuating HFD-induced metabolic dysregulation in GLUT10^G128E mice. Our results suggest that proper WAT development is essential for metabolic regulation later in life. Furthermore, when considering the usage of AA as a complementary nutrition for prevention and treatment of T2DM, individual differences in genetics and dietary patterns should be taken into account.

Keywords: Genetic predisposition; High-fat diet; Type 2 diabetes mellitus; Vitamin C; White adipose tissue.

Fig. 1

Ascorbic acid (AA) supplementation reduces HFD-induced weight gain. A Experimental overview. GLUT10^G128E or WT mating pairs, pregnant dams, nursing mothers, and weaned mice were supplied with drinking water supplemented with or without AA (3.3 g/L). The weaned mice were fed with normal diet (CD) for 2 weeks and then assigned to a CD or HFD from 5 to 20 weeks of age. B and C AA supplementation increases serum AA levels in mice at 3 weeks of age. Serum plasma AA levels were determined at 3 weeks of age (B) and 20 weeks of age (C) using the Ascorbic Acid Assay Kit. D and E AA supplementation reduces body weight gains in both WT and GLUT10^G128E mice. D Body weights were determined at indicated ages; n = 20 mice per group. E Fasting body weight at 20 weeks of age. The data are shown as mean ± SEM. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons. The compact letter display indicates significant differences in pairwise comparisons; groups with different letters are significantly different

Fig. 2

AA supplementation rescues HFD-induced metabolic dysregulation in GLUT10^G128E mice. Mice were treated as described in Fig. 1A. A HFD feeding significantly increased fasting blood glucose (FBG) levels in GLUT10^G128E mice at 20 weeks of age. FBG levels were determined at the indicated time point in CD- and HFD-fed WT and GLUT10^G128E mice. The data are shown as mean ± SEM. Statistical significance was determined by a two-tailed Student’s t-test. *P < 0.05. B–D AA supplementation improves metabolism-related readouts in HFD-fed GLUT10^G128E mice. Data were collected from mice at the conclusion of feeding (20 weeks of age). B Fasting glucose, C fasting HbA1c levels, and D fasting insulin levels. E and F AA supplementation had more pronounced effects on improving glucose tolerance and insulin sensitivity in HFD-fed GLUT10^G128E mice. E Glucose tolerance test (GTT) was performed on 16-week-old mice, and F insulin tolerance test (ITT) was performed on 18-week-old animals. Right panels in E and F show the areas under the GTT and ITT curves (AUC), respectively. The AUC were calculated using GraphPad Prism 7 software. n = 4 mice per group. B–F The data are shown as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Tukey’s test for multiple comparisons. The compact letter display indicates significant differences in pairwise comparisons; groups with different letters are significantly different

Fig. 3

AA supplementation reduces body fat composition and WAT weights in HFD-fed mice. Mice were treated as described in Fig. 1A. Data were collected from mice at the conclusion of feeding (20 weeks of age). A Representative photographs of mice, eWAT and sWAT from experimental mice. Each square on the green mat is 1 cm × 1 cm. B Body fat and C body lean compositions. D eWAT weights and E sWAT weights. The data are shown as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Tukey’s test for multiple comparisons. The compact letter display indicates significant differences in pairwise comparisons; groups with different letters are significantly different

Fig. 4

AA supplementation reduces HFD-induced eWAT inflammation and improves adipokine dysregulation in GLUT10^G128E mice. Mice were treated as described in Fig. 1A. Data were collected from mice at the conclusion of feeding (20 weeks of age). A AA supplementation reduces crown-like structures (CLSs) in eWAT of HFD-fed GLUT10^G128E mice. CLSs are a hallmark of eWAT inflammation. The eWAT sections were examined by H&E staining. Representing images are shown, and the red arrows point to a presumptive CLSs surrounding an adipocyte (left panel). The frequency of CLSs in eWAT sections were analyzed using ImageJ [54] and presented as CLS numbers per 100 adipocytes (right panel); n = 4 mice per group. B–E AA supplementation improves HFD-induced adipokine dysregulation in GLUT10^G128E mice. The serum levels of B adiponectin, C leptin, DIL-6, and E TNFα were determined. The data are shown as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Tukey’s test for multiple comparisons. The compact letter display indicates significant differences in pairwise comparisons; groups with different letters are significantly different

Fig. 5

AA supplementation reduces HFD-induced ectopic lipid accumulation in GLUT10^G128E mice. Mice were treated as described in Fig. 1A. Data were collected from mice at the conclusion of feeding (20 weeks of age). A–B AA supplementation reduces HFD-induced free fatty acid (FFA) levels and total cholesterol (TCHO) levels in GLUT10^G128E mice. A FFA levels and B TCHO levels in serum were determined. C–H AA supplementation rescues HFD-induced ectopic lipid accumulation in liver and iBAT. Representative photographs of C livers and D iBAT from experimental mice. Each square on the green mat is 1 cm × 1 cm. E Liver weights. F iBAT weights. H&E staining of G liver sections and H iBAT sections. The data are shown as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Tukey’s test for multiple comparisons. The compact letter display indicates significant differences in pairwise comparisons; groups with different letters are significantly different

Fig. 6

AA supplementation improves eWAT development in GLUT10^G128E mice. Mice were treated as described in Fig. 1A. Data were collected from pups at 3 weeks of age. A and B AA supplementation increases percentage of eWAT weight of GLUT10^G128E mice. A Body weight and B eWAT percent of total body weight were measured. C–E AA supplementation increases adipocytes size in eWAT of GLUT10^G128E mice. C Representative photographs of hematoxylin and eosin (H&E) staining of eWAT sections. The cross-sectional area of adipose cells in eWAT is presented as D average size of cells and E the percentage of cells in the given size range. In D and E, n = 6 mice per group; more than 1000 adipocytes were analyzed in each mouse. F AA supplementation reduces Pref-1 protein levels in eWAT of GLUT10^G128E mice. The protein levels of Pref-1 and PPARγ 1 in eWAT were analyzed by Western blotting. Protein samples from 6 mice per group were pooled. The protein levels were quantified, normalized to β-actin levels, and compared to WT no-AA-treated controls. The data are shown as mean ± SEM from triple repeats of Western blotting experiments of the pooled samples. Statistical significance was determined by one-way ANOVA followed by Tukey’s test for multiple comparisons. The compact letter display indicates significant differences in pairwise comparisons; groups with different letters are significantly different

Fig. 7

Model of AA-mediated attenuation of HFD-induced metabolic dysregulation in GLUT10^G128E mice. AA supplementation in pregnant dams, nursing mothers, and weaned mice rescues eWAT development in GLUT10^G128E pups and attenuates later HFD-induced eWAT inflammation and metabolic dysregulation in GLUT10^G128E mice